Tumor specific monoclonal antibodies

ABSTRACT

Monoclonal antibodies produced by hybridoma or transformed B-cell lines deriveed from B-cells of cancer patients actively immunized with autologous tumor antigen. These monoclonal antibodies can be used in both diagnostic procedures and therapy for human cancers.

This application is a continuation-in-part of U.S. patent applicationSer. No. 06/575,533,filed Jan. 31st, 1984 now abandoned.

DESCRIPTION OF THE INVENTION

This invention relates to monoclonal antibodies produced by hybridoma ortransformed B-cell lines derived from B-cells of cancer patientsactively immunized with autologous tumor antigen. These monoclonalantibodies can be used in both diagnostic procedures and therapy forhuman cancers. This invention also relates to diagnostic procedures andtherapeutic approaches using these monoclonal antibodies.

BACKGROUND OF THE INVENTION

This invention relates to new human monoclonal antibodies which reactspecifically with antigens associated with particular cancers and tohybridoma and transformed B-cell lines for their production derived fromperipheral blood B-cells of actively immunized patients. This inventionalso relates to methods having general applicability to all solidcancers for preparing hybridomas and monoclonal antibodies and todiagnostic procedures and cancer therapy using these monoclonalantibodies.

Currently available treatments for cancer, particularly radiationtherapy and chemotherapy, are based upon the rationale that cancer cellsare relatively more sensitive to these treatments than normal cells.However, severe toxicity for normal tissues imposes major limitations tothese therapies. In contrast, antibody molecules exhibit exquisitespecificity for their antigens. Researchers have therefore sought toisolate antibodies specific for cancer cells as the "long-sought `magicbullet` for cancer therapy" Marx, (Science, 1982, 216:283).

Antibodies are protein molecules normally synthesized by the B-celllymphocytes produced by bone marrow and carried in the blod stream. Forany antigen entering the body, i.e., any foreign molecule from a simpleorganic chemical to a complex protein, antibodies are produced whichrecognize and attach to that particular chemical structure. The uniquechemical structure on the antigen to which a particular antibody canbind is referred to as an antigenic determinant or epitope. B-celllymphocytes in the body, referred to as B-cells, lymphocytes, orleukocytes, exist as hundreds of millions of different geneticallyprogrammed cells, each producing an antibody specific for a differentdeterminant. An antigen, which stimulates antibody production, can haveseveral determinants on its surface. On encountering an antigen, aB-cell carrying on its surface an antibody specific for a determinant onthat antigen will replicate. This clonal expansion results in manydaughter cells which secrete that antibody into the blood stream.

Because of the specificity of antibodies in recognizing and binding toantigens, it was desired to produce antibodies in quantity which arespecific for a single determinant, thus binding only to antigens ortissues having that particular determinant.

B-cells do not grow in a continuous culture unless they have beenaltered by hybridization with an "immortal" cell or by being transformedwith either viral or tumor DNA. Kohler and Milstein (Nature, 1975, 256:495) demonstrated that hybrid cells could be prepared by somatic cellfusion between lymphocytes and myeloma cells which grow in culture andproduce an antibody specific for a single determinant. These hybrids arereferred to as "hybridoma cells." Hybridoma cells are prepared by fusinglymphocytes, which have been activated to produce a particular antibody,with myeloma cells. When cultured, hybridomas produce antibodiesspecific for a single determinant on a particular antigen. Suchantibodies are referred to as "monoclonal antibodies."

Monoclonal antibodies may also be produced by B-lymphocyte cell linesthat have been spontaneously transformed, either prior to or subsequentto being placed in culture. These cells, in distinction to hybridomacells, possess a normal human diploid number (46) of chromosomes. Thisinvention permits the isolation of both hybridomas and transformedB-cell lines that produce monoclonal antibodies. For sake of simplicity,both cell types will be referred to as monoclonal antibody producingcells below.

Monoclonal antibodies are synthesized in pure form by a monoclonalantibody producing cell cultures uncontaminated by otherimmunoglobulins. With such a cell culture, it is possible to producevirtually unlimited quantities of an antibody that is specific for onedeterminant on a particular antigen.

It has been believed that if antibodies specific for particular cancercells were available, they could be used in various methods of treatmentand diagnosis. Such antibodies could inactivate or kill particular tumorcells merely by attaching to the cell at the determinant for which theyare specific. Alternatively, these antibodies may bind to the surface ofeffector lymphocytes or macrophages, converting them into tumorantigen-specific killer cells.

Monoclonal antibodies can also increase the specificity ofchemotherapeutic drugs, toxins and radioactive isotopes, thus increasingtheir efficacy while decreasing their toxicity. A monoclonal antibodycan be conjugated with a toxin, radionuclide or chemotherapeutic drug;this conjugated antibody may be simplistically viewed as a guidedmissile with the antibody as the guidance system and the drug as thewarhead. In addition, antibodies conjugated with radionuclides ormetallic tracers can be used for proton emission (PET) and nuclearmagnetic resonance (NMR) imaging for in vivo diagnosis and localizationof metastases. The antibodies can also be used for detecting thepresence of tumor antigens in blood, as a diagnostic and/or prognostictest for cancer. Also, monoclonal antibodies can be used to isolate thetumor antigens for potential use in a standardized vaccine.

The existence of antigens associated with animal tumors was documentedin the last century, and the antigenic character of human cancers hasbeen well established, primarily through recent studies with monoclonalantibodies. However, until the research which resulted in thisinvention, few cancer antigens have actually been characterized inmolecular terms and only one group of antigenic determinants associatedwith human cancers, immunoglobulin idiotypes of B-cell tumors, has beendescribed as being uniquely tumor-specific, i.e., occurring with a highfrequency on tumor cells and not occurring to any significant degree onnormal tissues (Oldham and Smalley, J. Biol. Response Modifiers, 1983;Stratte et al., J. Biol. Response Modifiers, volume 1, 1982).

DESCRIPTION OF THE PRIOR ART

Past attempts at deriving monoclonal antibodies specific for humancancers have taken two routes with respect to B-cells: (1) B-cells havebeen extracted from spleens of mice that were immunized against humantumors, U.S. Pat. No. 4,172,124; and (2) human B-cells have beenextracted from either peripheral blood or from lymph nodes drainingtumors in cancer patients. Neither approch has yielded satisfactoryresults.

Mice immunized against human tumors have too broad a reactivity. Thatis, most of the mouse monoclonal antibodies generated react with humanantigens present on normal as well as on tumor tissue. An antibody thatreacts only with tumor cells is very difficult to select from among thelarge variety of antibodies produced. For example, 20,000 hybridomasderived from mice immunized with human small-cell lung carcinoma werescreened for reactivity with tumor cells Marx, (Science, 1982, 216:283).In contrast to a very low frequency (<0.4%) observed by this researchgroup, the present invention results in up to 16% of the hybridomasderived from immunized colon patients producing monoclonal antibodiesthat react specifically with tumor cells. In addition, monoclonalantibodies derived from mouse B-cells have limited potential forapplication in cancer therapy. After repeated administration they tendto stimulate the human immune system to produce "anti-mouse" antibodieswhich, in clinical trials, have been shown to neutralize the activity ofmouse monoclonal antibodies. The use of our human monoclonal antibodiescan circumvent these difficulties.

Another apparent difference between human and mouse monoclonalantibodies is their patterns of labeling. Previous studies with mouseantibodies have demonstrated that there is often a heterogenous labelingof cells within tumor sections. This pattern of reactivity has beenattributed by some authors to antigenic heterogeneity of tumor cells(Hand et al., Cancer Research, 43:728-735, 1983). In contrast, the humanmonoclonal antibodies developed by our strategy were homogeneous interms of their reactivity to tumors to which they did react. A plausibleexplanation for the heterogenous staining of mouse monoclonal antibodiesis that it is a reflection of the murine immune recognition of phase- orcell-cycle-specific differentiation antigens abundant on the tumor cellsrather than putative tumor associated antigens. It is not unreasonableto expect that when one immunizes mice with human tumor cells, therewould be substantial antigenic competition resulting in the moreabundant and more predominant tissue-type and differentiation antigenssuccessfully competing with relatively minor tumor associated antigensfor immune responsiveness by the host. Thus, autologous immunization ofman may result in the elicitation of antibodies against the group ofantigens normally poorly immunogenic in mice. This evidence suggeststhat humans and mice may respond to different tumor antigens. In concertwith this hypothesis is our finding that none of the 36 human monoclonalantibodies we produced appear to react with carcinoembryonic antigen(CEA), an antigen frequently recognized by murine monoclonal antibodiesmade against human tumor cells.

The majority of past attempts to develop human monoclonal antibodieshave used B-cells extracted from either peripheral blood or lymph nodesfrom patients bearing tumors. It was believed that the presence of theantigenic tumor would cause a tumor-bearing individual to mount animmune response against his tumor and produce specifically immuneB-cells. Thus, B-cells were taken from lymph nodes draining tumors incancer patients or from circulating lymphocytes found in peripheralblood. However, prior to the present invention, there has been limitedsuccess in creating tumor-specific monoclonal antibodies.

The major problem in creating monoclonal antibodies specific for humantumor antigens has been the inability to find a source of specificallyimmune B-cells Marx, (Science, 1982, 216:285). In humans, the initialfoci of cancer cells tend to grow over long periods of time, from 1% to10% of the human lifespan, before there is any palpable clinicalevidence of the disease. By this time patients are immunologicallyhyporesponsive to their tumors, or possibly immunologically tolerant.Thus, prior to the present invention, human monoclonal antibodiesreactive with tumor cells could not reproducibly be obtained.Furthermore, of the small number of human monoclonal antibodies obtainedfrom cancer patients, very few reacted with determinants found on thesurface of tumor cells, but rather with intracellular determinants (R.J. Cote et al., PNAS, 1983, 80:2026). The present invention permits thedevelopment of monoclonal antibodies reactive with surface antigens; arequisite activity for tumor imaging and therapy.

SUMMARY OF THE INVENTION

One object of the present invention was to develop monoclonal antibodiesreactive with tumor-specific antigens that induce an immune response inpatients having particular cancers. A valid in vivo assay for theimmunogenicity of tumor-specific antigens in tumor immunized patients isby delayed cutaneous hypersensitivity. Such antibodies provide a meansfor detecting and diagnosing tumors. A second objective of thisinvention was to obtain monoclonal antibodies which would be effectivein treating patients with particular types of cancer.

We have developed a new and more effective approach for obtainingmonoclonal antibodies by using peripheral blood B-cells from patientsimmunized with cells from their own tumors in a specific vaccinepreparations. To achieve active specific immunotherapy, patients wereimmunized with autochthonous tumor cells, that is, cells from their owntumors. This approach was taken based on our theory that tumor cellsexpress tumor-specific antigens.

Animal model studies have supported the concept that antigens not foundin normal adult tissues are frequently found in tumors, and that theimmunogenicity of these tumor cells can be expressed, and even enhanced,in both normal and tumor-bearing hosts. These experimental resultsvalidated the rationale of active specific immunotherapy in humanneoplasia.

Humans mounting an objective immune response against tumor cells werespecifically found to be a good source of activated B-cells. Theperipheral blood of patients who had been actively immunized againsttheir own tumors was shown in clinical trials to be an abundant sourceof such activated B-cells.

It was demonstrated in clinical studies that an objective immuneresponse is generated on treating patients having the particular cancerby skin testing, i.e., delayed cutaneous hypersensitivity (DCH).Immunized patients showed delayed cutaneous hypersensitivity to theirown colorectal cancers. In addition, the monoclonal antibodies developedfrom the immunized patients' B-cells reacted with tumors of the samehistological type in other patients. These results indicate that thepatient's humoral immune response, production of antibodies, is directedagainst colorectal cancer generally and is not unique to the immunizedpatient's own tumor. This general response is especially important forthe development of a standardized vaccine.

The treatment also proved to be highly beneficial. Forty-two monthsafter the immunization of the first patients there has been an objectiveand significant improvement in the patients with respect to duration ofthe disease-free period following surgery, and the survival data areencouraging. Only 3 of 20 treated patients had recurrences and none havedied. Comparatively, 9 of 20 patients in a control group had recurrencesand four have died.

The generation of B-cells which produce antibodies having reactivityspecific for tumor cell antigens, particularly cell surface antigens asin the majority of cases, is an advantageous result that wasspeculative, at best, when the immunization studies were begun. Only theimmunization treatment was observed and measured during the animalstudies on which the human immunization procedures were based, not theproduction of tumor specific antibodies.

The general immune response accompanied by an improvement in thesubject's condition was indicative of a cellular response in whichmacrophages and T-cells become activated in the presence of tumor cellantigens and destroy the tumor cells. Although an antibody responsewould predictably be triggered by immunization under most circumstances,the time course of the antibody response and the cellular response wouldin most instances be different. Moreover, the fact that the patientswere being immunized with autologous tumor cells, and the experience ofprevious investigators that little or no antibody production istriggered by a patient's own tumor, made our discovery that B-cellswhich produce tumor specific antibodies are generated after immunizationan unexpected beneficial result.

Some cellular and humoral immune responses can occur independently ofeach other. For example, it is possible to mount a humoral response inthe absence of demonstrable cellular immunity. Conversely, potentcellular immunity, particularly delayed cutaneous hypersensitivity(DCH), may develop despite a minimal antibody response. It wassurprising, therefore, for the subjects who showed a positive responseto active immunotherapy to have been excellent sources of B-cellsproducing tumor specific antibodies, particularly cell surfaceantibodies.

A third objective of this invention was to prepare a standardizedvaccine for use in detecting and treating specific cancers in thegeneral population which did not require the custom preparation of a newimmunogen suitable for each individual patient. Without a standardizedvaccine, only a vaccine prepared for each individual patient from hisown tumor tissue could be used for therapy, and only known cancers couldhave been treated on a limited scale in large institutions. It would nothave been possible to make individual preparations for treating theapproximately 139,000 cases of colorectal cancer that are discovered inthe United States every year.

This invention comprises the preparation of successful vaccines foractive specific immunization, procedures for extracting immunizedB-cells, the production of monoclonal antibody producing cells and theproduction of monoclonal antibodies. Malignant tumors are digested usingenzyme preparations. The cells obtained are treated to yield anon-tumorigenic tumor cell preparation having the requisite cellviability, which is injected as a vaccine into the subject from whichthe tumor was obtained. Peripheral blood B-cells are obtained from theinoculated subject after a predetermined interval and are used toprepare monoclonal antibody producing cells by fusing with myelomacells, after which the fused cells are screened for the synthesis ofimmunoglobulin. Cells that synthesize immunoglobulin are tested forproduction of antibodies which react with antigens characteristic of themalignant tissue. Those selected are cultured to produce monoclonalantibodies that react with the particular type of tumor with which thesubject was afflicted.

Mouse myeloma cells grown in culture were used to prepare hybridomas inthe research which led to this invention. However, as the problems withdeveloping easy-to-grow human myeloma cell lines that do not produceantibodies of their own are solved, human myelomas will be preferred forpreparing the hybridomas of this invention.

DETAILED DESCRIPTION OF THE INVENTION

The key aspects of this invention are:

(1) Criteria for successful vaccines for active specific immunization:

Tumor cells, whole cells enzymatically dissociated from tissue,cryopreserved and X-irradiated for non-tumorigenicity.

Adjuvant, an immunomodulator that is capable of inducing immunogenecityto the tumor cell preparation.

Components and administration, including ratio of adjuvant to tumorcells, optimum doses of tumor cells, and regimen of vaccination.

Patient, regional lymph nodes draining the vaccination site must bepresent during the first 21 days after vaccination.

(2) Procedures and timing for the extraction of immunized B-cells fromthe patients.

(3) Procedures for the production of hybridomas and transformedlymphocytes and production of monoclonal antibodies.

We have successfully digested solid human malignancies using variousenzyme preparations. The tumor dissociations were evaluated for yield oftumor cells per gram of tissue, cell types recovered, cell viability,cell size, and sterility. The criteria for successful vaccines foractive specific therapy are shown in Table 1.

Tumor tissue was obtained from patients suffering from the particularsolid cancer for which monoclonal antibodies were to be prepared. Thetumor tissue was surgically removed from the patient, separated from anynon-tumor tissue, and cut into small pieces. We found it satisfactory tocut the tumor tissue into fragments 2-3 mm in diameter. The tumorfragments were then digested to free individual tumor cells byincubation in an enzyme solution.

After digestion, the freed cells were pooled and counted, and cellviability was assessed. The trypan blue exclusion test was found to bean acceptable measure of cell viability. The tumor cells were thencryopreserved and stored in liquid nitrogen.

The vaccine was prepared for injection by rapidly thawing cryopreservedcells, diluting the cells, washing with HBSS, resuspending, counting,and assessing viability.

Viable tumor cells were irradiated to render them non-tumorigenic. Wefound that irradiation with 4020 rads/min for a total of 20,000 radsresulted in non-tumorigenic but viable cells. The volume of the cellsuspension in HBSS was adjusted such that 10⁷ viable cells remained inthe tube. The cells were centrifuged, the supernatant was removed, and10⁷ viable BCG were added in a volume of 0.1 ml. Hank's Balanced SaltSolution (HBSS) was added in sufficient quantity for a final volume of0.2 ml. A third vaccine was similarly prepared, omitting the BCG.

Immunization of Patients

Patients afflicted with the particular solid cancer for which antibodieswere to be generated were immunized by intradermal inoculation with thetumor cell vaccine. 10⁷ viable tumor cells admixed with BCG were usedfor the first two vaccinations and 10⁷ tumor cells alone were used forthe third vaccination. Scheduling each vaccination one week apart wasfound to be a successful procedure for inducing antibody production bythe patients' peripheral blood lymphocytes.

Collection of Immunized B-Cells

Venous blood was collected from the immunized patients one week aftereach vaccination. Peripheral blood lymphocytes (PBL) were separated fromthe collected blood for use in hybridoma production.

Separation of lymphocytes from the blood was accomplished using twodifferent methods. The first comprised dilution with calcium andmagnesium-free HBSS, layering on lymphocyte separation medium,centrifuging, and removing cells at the interface. These cells werediluted with HBSS and pelleted. The lymphocytes were then resuspended inserum-free Hepes-buffered Dulbecco's MEM (DMEM), counted, and assayedfor viability (GIBCO Biologics, Grand Island, N. Y.).

An alternative method that was used to recover peripheral bloodlymphocytes (PBLs) enriched for B-cells comprised the removal ofT-lymphocytes by rosetting with 2-aminoethylisothiouronium bromidehydrobromide (AET) treated sheep erythrocytes. Treated erythrocytes weremixed with peripheral blood lymphocytes, pelleted by centrifugation, andthe pellet incubated on ice. After resuspension, layering overlymphocyte separation medium (LSM, Litton Bionetics), and centrifugationof the rosetted cells, the T-cell depleted PBLs were collected at theinterface, washed, and pelleted. The PBLs enriched for B-cells were thenused for hybridoma generation after counting and viabilitydetermination.

Preparation of Human Hybridomas for the Production of Anti-TumorMonoclonal Antibodies

Peripheral blood lymphocytes (PBLs) and cultured myeloma cells weremixed together, pelleted, and resuspended in a serum-free medium.Polyethylene glycol (PEG) was added, the cells pelleted and resuspendedin HT medium (DMEM containing 20% fetal bovine serum, hypoxanthine andthymidine) and distributed into microtiter wells. Twenty-four hourslater, HAT medium (HT medium containing aminopterin) was added to eachwell, with one-half of the medium being replaced every three days. Aftermaintenance in HAT medium for 14 days, the cells were maintained on HTmedium for an additional two weeks, after which the cells were grown ona DMEM medium containing 20% fetal bovine serum.

The hybridomas were pre-screened for the synthesis of humanimmunoglobulin using the standard enzyme immunoassay. Hybridomassynthesizing human immunoglobulin in sufficient amounts were tested ontissues. Particular tissue samples were incubated with hybridomasupernatant fluids. Supernatants which demonstrated reactivity withparticular tumor tissues indicated that hybridoma cells in the wellsfrom which the particular supernatants were drawn producedtumor-specific antibodies. If the same supernatants failed to show areaction with samples of normal tissue after extensive screenings, thehybridomas in that particular well were considered tumor-specific. Thesetumor-specific supernatants were further tested against carcinoembryonicantigen (CEA) to be sure of their narrow specificity.

In addition to hybridoma cells which produced tumor-specific antibodies,transformed human B-cells (diploid cells) were also prepared by theseprocedures which also produced tumor-specific antibodies. Thetransformed B-cells were detected in the same way as tumor-specificantibody-producing hybridomas. Thus, well supernatants which testedpositively for reactions with tumor tissue and negatively for reactionswith normal tissue and with CEA contained either hybridomas ortransformed B-cells. The two types of cells were differentiated byobserving that the transformed B-cells contained 46 human chromosomes,whereas the hybridomas contained many more chromosomes, not all of whichwere of the human type.

The mechanism by which B-cells become transformed during the abovedescribed procedures has not been precisely determined.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1A

Chromosome spread of a cell with growth characteristics typical ofhybridomas (X1600). LiCo 21B27 was incubated with colcemid (0.05 μg/ml)for two hours and treated with hypertonic (0.075M) KCl for threeminutes. The cells were fixed with methanol-acetic acid (3:1), droppedonto microscope slides, air-dried and stained with Giemsa. Both humanand mouse chromosomes are present.

FIG. 1B

Phase photomicrograph of a clusterforming monoclonal antibody (LiCo18-15) producing cell line (X270). Note the aggregation and irregularshape of the cells.

FIG. 1C

G-banded chromosome spread of the cell line shown in FIG. 1D (X1360).Note the absence of mouse chromosomes. The cells were incubated withcolcemid (0.01 μg/ml) overnight. The chromosome spreads were prepared asdescribed above. The unstained slide was aged for 10 days. Thechromosomes were treated with trypsin (0.19% for 30 seconds at roomtemperature), dehydrated with ethanol and stained with Giemsa.

FIG. 1D

Formalin-fixed (10%) paraffin-embedded section of a colon carcinomareacted with LiCo 16-88 (4 μg/ ml IgM×380). Both surface-like andcytoplasmic labeling are seen. The deparaffinized section was blocked(20 min. at room temperature) with phosphatebuffered saline (PBS) (pH7.3) containing 0.75 M L-lysine and 1% bovine serum albumin and thenincubated with LiCo 16-88 overnight at 4° C. After washing with PBS thesection was incubated (60 min. at 37° C.) with affinity-purifiedperoxidas-labeled goat antibody to human immunoglobulins (IgG+IgA+IgM),washed and then reacted (15 min. at room temperature) withdiaminobenzidine (0.5 mg/ml) in PBS (pH 7.6) containing 0.1% H₂ O₂.After counterstaining with hematoxylin, the section was dehydrated,cleared and mounted with permount.

FIG. 1E

Colon tumor as in FIG. 1D, reacted with normal human IgM (4 μg/ml)(x380). No staining is observed.

FIG. 1F

Cryostat section of a colon tumor stained by LiCo 16-88 (x640). Note theintense label of the periphery of the tumor cells (arrows). The sectionwas air dried and stored at -30° C. This section was post-fixed (20 min.at 4° C.) with PLP in PBS and processed as described in FIG. 1D, exceptthat peroxidase-labeled goat antibody specific to human μ chains wasused.

FIG. 1G

Cryostat sections of the colon tumor seen in FIG. 1F reacted with normalhuman immunoglobulin (x640). No labeling of the tumor cells is seen.

FIG. 1H

Cytospin preparation of air-dried unfixed SW1463 cells stained by LiCo16-88 (4 μg/ml) (x280). The colon tumor cell line was harvested withethylenediaminetetraacetic acid (EDTA) (0.02%), washed and suspended inmedium containing 1% bovine serum albumin. Cells (2×10⁴ in 0.1 ml) werepelleted onto the glass slides in a cytocentrifuge, air dried and storedat -30° C. Cells were incubated with monoclonal antibody (1 hr. at roomtemperature and then overnight at 4° C.), washed and then processed asdescribed above.

FIG. 2

Distribution of antigens in paraffin sections of colorectal tumors.Shaded area indicates positive indirect immunoperoxidase staining of 15tumors by 10 human monoclonal antibodies.

FIG. 3

Two monoclonal antibodies react with most colorectal tumors. Thereactivity of two monoclonal antibodies to paraffin sections of 15colorectal tumors and air-dried cytospin preparations of dissociatedtumors from 9 patients are compared. Shaded area indicates positiveindirect immunoperoxidase staining.

FIG. 4

Follow-up of all control and immunized patients in active specificimmunotherapy clinical trials according to site and pathologic stage.

FIG. 5A

Disease-free status of all patients.

FIG. 5B

Survival status of all patients.

FIG. 6A

Disease-free status of patients with positive regional lymph nodes(Astler-Coller C).

FIG. 6B

Survival status of patients with positive regional lymph nodes(Astler-Coller C).

Example I: Preparation of Sensitized B-Cells A. Patient Selection

Patients undergoing surgical resection of colon or rectal cancers wereselected for a randomized trial of active-specific immunotherapy.Randomization was done with stratification according to pathologic stageand tumor was obtained from all patients who met the clinical criteria.Candidates for the study were colorectal cancer patients with noprevious history of cancer, who had received no prior chemotherapy orradiation therapy, and who were in suitable medical condition to complywith the outpatient treatment protocol. Patients eligible for the trialwere those with tumor extending through the bowel wall (Astler-CollerB2), positive lymph nodes (stages C1, C2) or patients with metastaticdisease (stage D). Within these classifications, patients were randomlyselected for participation in treatment and nontreatment groups.Randomization cards were computer generated and sequentially drawn fromeach category postoperatively.

B. Tumor Acquisition

After surgical resection the bowel specimen was taken immediately to thehospital pathology department and opened under sterile conditions. Tumortissue was excised, placed in sterile tubes containing Hank's BalancedSalt Solution (HBSS) containing 50 μg gentamicin per ml and carriedimmediately on ice to the laboratory for processing and freezing.

C. Dissociation of Solid Tumor and Colon Mucosa

The tissue dissociation procedure of Peters et al. (Cancer Research,39:1353-1360, 1979) was employed using sterile techniques throughoutunder a laminar flow hood. Tumor tissue was rinsed three times in thecentrifuge tube with HBSS and gentamicin and transferred to a petri dishon ice. Scalpel dissection removed extraneous tissue and the tumor wasminced into pieces approximately 2 to 3 mm in diameter. Tissue fragmentswere placed in a 75 ml flask with 20-40 ml of 0.14% (200 units/ml)Collagenase Type 1 (Sigma C-0130) and 0.1% (500 Kunitz units/ml)deoxyribonuclease type 1 (Sigma D-0876) (DNAase 1, Sigma D-0876)prewarmed to 37° C. Flasks were placed in a 37° C. waterbath withsubmersible magnetic stirrers at a speed which caused tumbling, but notfoaming. After a 30-minute incubation, free cells were decanted throughthree layers of sterile medium-wet nylon mesh (166 t: Martin Supply Co.,Baltimore, Md.) into a 50 ml centrifuge tube. The cells were centrifugedat 1200 rpm (250× g) in a refrigerated centrifuge for 10 minutes. Thesupernatant was poured off and the cells were resuspended in 5-10 ml ofDNAase (0.1% in HBSS) and held at 37° C. for 5-10 minutes. The tube wasfilled with HBSS, washed by centrifugation, resuspended to 15 ml in HBSSand held on ice. The procedure was repeated until sufficient cells wereobtained, usually three times for tumor cells. Cells from the differentdigests were then pooled, counted, and cell viability assessed by thetrypan blue exclusion test. The cells were centrifuged for a final washprior to cryopreservation.

D. Cryopreservation

Optimal cryopreservation was a primary concern. For vaccine preparation,the dissociated tumor cells were adjusted to 5-8×10⁷ /ml in HBSS andadded in equal volume to chilled 2× freezing medium containing 15%dimethylsulfoxide (DMSO) and 4% human serum albumin (HSA). The finalsuspension of 2 to 4×10⁷ cells/ml were placed in 1.2 ml Nunc freezervials. For DCH cell testing the procedure was the same except that noHSA was used. In both cases, in preparation for freezing, the Nunc vialswere transferred on ice to a Cryo-Med model 990 Biological Freezer witha model 700 Controller and a model 500 Temperature Recorder forcontrolled-rate freezing. Care was taken that the temperature of theindividual vials, including the monitor vial, was uniform at thebeginning of the freezing process. vials were cooled at a controlledrate of -1° C./min to a final temperature of -80° C. The vials weretransferred in liquid nitrogen to liquid nitrogen storage.

E. Clinical Protocol

Patients with tumors of the appropriate pathologic stages wererandomized to receive either the autologous tumor cell-BCG vaccine or tohave no further therapy. The stage D patients all received5-fluorouracil chemotherapy and all patients with lesions below theperitoneal reflection (rectal cancer) received 5040 rads of pelvicX-irradiation two weeks after immunotherapy was completed. The vaccineswere started at 4-5 weeks after tumor resection to allow sufficient timefor recovery of immunologic suppression induced by anesthesia andsurgery. At 3-4 weeks after resection, both control and treatmentpatients were skin tested with standard recall antigens as well asgraded doses of their autologous tumor cells. Recall antigens used were:Mumps skin test antigen, USP, Eli Lilly, Indianapolis, Ind.; Aplisol,PPD, (Tuberculin Purified Protein Derivative), Parke-Davis, Detroit,Mich.; Trichophyton, diluted 1:30, Center Laboratories, Port Washington,N.Y.; and Candida albicans diluted 1:100, Center Laboratories, PortWashington, N.Y., 0.1 ml of each was placed intradermally on the forearmand examined for erythema and induration at 24 and 48 hours.

Patents selected for treatment protocol received 3 weekly intradermalvaccine injections consisting of 10⁷ irradiated, autologous tumor cellsand 10⁷ BCG in the first 2 vaccines with 10⁷ tumor cells alone in thefinal. Fresh-frozen Tice BCG, supplied by Dr. Ray Crispen, University ofIllinois Medical Center, Chicago, Ill., was stored at -70° C. The firstvaccine was placed on the left anterior thigh approximately 10 cm belowthe groin crease, the second in a comparable location on the right thighand the third in the right deltoid area.

F. Preparation of vaccine

On the day of the first and second vaccinations, the vial was rapidlythawed in a 37° C. waterbath, tumor cells were diluted slowly to 15 mlin HBSS, washed once by centrifugation at 1200 rpm and resuspended to 15ml in HBSS. Cell counts and viability determinations were made using thetrypan blue exclusion test. Viability ranged between 70 and 90%, with amean of 80%. The cells were washed once by centrifugation at 1200 rpmand resuspended to 15 ml in HBSS. The suspension of tumor cells wasplaced on ice and irradiated at 4020 rads/min for a total of 20,000rads. The volume of the cell suspension was adjusted such that 10⁷viable tumor cells remained in the tube (1.3×10⁷ viable cells areincluded to allow for cell loss in tubes and syringes, and for thepossibility of approximately 20% misidentification of lymphoid cells).The cells were centrifuged, the supernatant removed and 10⁷ BCG wereadded in a volume of 0.1 ml. HBSS was added in sufficient quantity for afinal volume of 0.2 ml. The third vaccine was similarly prepared,omitting the BCG.

The vaccine suspension was drawn up through a 20 gauge needle into a 1.0ml tuberculin syringe. The 20 gauge needle was replaced with a 27 gaugeneedle for the intradermal injection, and the syringe was placed on icefor transport to the clinic.

The patients were observed closely after each vaccine for erytherma andinduration at the site of injections, fever, lymphadenopathy or anyadverse reactions. The first two vaccine sites ulcerated after 2-3 weeksbut always healed within 10 to 12 weeks.

G. Results of Immunization Reactivity to Standard Recall Antigens

All patients were reactive initially to at least one of the standardrecall antigens. Two of the 29 were reactive to candida, 26 of 29 werereactive to mumps, 16 of 29 were reactive to PPD and 3 of 29 reacted totrichophyton. There was no significant change in reactivity in thefollowup period except that all but two of the immunized patentsconverted to PPD positivity.

H. Delayed Cutaneous Hypersensitivity (DCH) to Tumor Cells

The delayed cutaneous hypersensitivity reaction to 10⁶ autologous tumorcells in 24 immunized and 11 nonimmunized control patients is shown inTable 2. A 48-hour induration measurement of greater than 5 mm wasconsidered positive. Four of 24 patients (17%) had a positive DCH to 10⁶tumor cells prior to the course of immunization. This was notsignificantly different from the one of 11 patients (9%) testingpositive in the nonimmunized control group. Of significance (p<0.01) allof the initially four positive responders and 12 of the negativeresponders in the immunization group boosted to greater DCH reactivityfollowing a course of immunotherapy (67% became positive). Seven ofthese patients have been tested at one year, with three maintaining apositive response. Only three of the 16 objectively immunized patientsdemonstrated a positive DCH response to 10⁵ tumor cells at 6 weeks, withnone showing a response to 10⁴ cells.

Example II: Production of Hybridomas for Human Monoclonal Antibodies A.Removal and Processing of Immunized B-Cells from Patients

Patients were bled at the time of the second immunization, one weekafter the first immunization, and at the time of the third vaccination,one week after the second immunization. Venous blood was collectedasceptically in the presence of preserative-free heparin (O'Neill, Jonesand Feldman, St. Louis, Mo.) at a final concentration of 17 units/ml.The blood was maintained at room temperature and transported to thelaboratory expeditiously, within a few hours of collection.

The blood, diluted 1:2 with calcium and magnesium-free HBSS, was layered(4 ml) over 3 ml of lymphocyte separation medium (LSM, Litton Bionetics)and centrifuged in a 15 ml centrifuge tube for 30 minutes at 400×g. Thecells at the interface were removed, diluted with three times theirvolume of HBSS and pelleted (1000 rpm for 10 minutes). The peripheralblood lymphocytes (PBL) were resuspended in 10 ml of serum free Hepesbuffered Dulbecco's MEM (DMEM), counted and viability determined.

An alternative method was also used to recover immunized B-cells. TheT-lymphocytes were removed by rosetting with AET-treated sheeperythrocytes. Sheep erythrocytes (in Alsever's solution) were washedthree time with balanced salt solution (BSS) and incubated at 37° C. for20 minutes with four times the packed cell volume with 0.14 M AET(Sigma). The treated cells were then washed three times with HBSS andresuspended to a 10% suspension. The treated erythrocytes were layeredover LSM, centrifuged at 2500 rpm and the pellet collected. Followingthree washes with HBSS, the sheep erythrocytes were resuspended to a 10%suspension in undiluted fetal bovine serum and used within two weeks.The PBL (up to 80 million cells) were mixed with 1 ml of AET-treatedsheep erythrocytes and pelleted at 1000 rpm for 10 minutes at 4° C. Thepellet was incubated on ice for 45 minutes, gently resuspended with awide bore pipette and layered over 3 ml LSM. The rosetted cells werecentrifuged at 400×g for 40 minutes at room temperature. The T-celldepleted PBLs were collected at the interface, washed with three timesthe volume HBSS, and pelleted. Following counting and viabilitydetermination, the PBLs enriched for B-cells were then used forhybridoma generation.

B. Generation of Human Hybridomas

Mouse myeloma cells (NS-1) were grown in the presence of 8-azaguanine(20 μg/ml). Three days before fusion, the cells were pelleted andpassaged in medium free of 8-azaguanine. The cells were passaged againthe day before fusion to maintain them in log phase. The myeloma cellswere washed once with serum-free medium, counted, and viabilitydetermined. The PBL and myeloma cells were mixed at a ratio of 3:1 andpelleted together at 1000 rpm for 10 minutes. All supernatant fluid wasremoved and the cell pellet resuspended in less than 100 μl ofserum-free medium. One ml of polyethylene glycol (50% w/v) prewarmed to37° C. was added dropwise to the cell pellet over the course of oneminute with constant agitation of the tube. Twice the previous volume ofpre-warmed serum-free medium was added to the cell suspension over thecourse of one minute until the 50 ml tube was filled. The cells werepelleted at 800 rpm for 15 minutes. The cells were gently resuspended inHT medium (DMEM containing 20% fetal bovine serum, hypoxanthine 13.6μg/ml and thymidine 3.9 μg/ml) at a concentration of 2.5×10⁶ cells/ml(pre-fusion count) and 100 μl was added to each microtiter well.Twenty-four hours later, 100 μl of HAT medium (HT medium containing 0.18μg/ml aminopterin) was added to each well. Half of the medium wasreplaced every three days with fresh HAT medium. After maintenance inHAT medium for 14 days, the cells were maintained on HT medium for anadditional two weeks, at which time the cells were grown on a DMEMmedium containing 20% fetal bovine serum.

Alternatively, co-cultivation of PBL with myeloma cells may be used togenerate transformed diploid B-cells. PBL and myeloma cells were mixed(at a ratio of 3:1), pelleted at 800 rpm and selected in HAT medium, asdescribed above.

C. Screening of Hybridomas

The hybridomas were first quantified and isotyped by a captureenzyme-linked immunoassay (ELISA) for the synthesis of humanimmunoglobulin (IgA, IgG and IgM). The standard Bio-EnzaBead™ method wasutilized, which is sensitive in the range of 10-300 ng/ml. The hybridomasupernatant fluids were diluted 1:30 with an effective range of 0.3-9μg/ml. Only hybridomas that synthesized human immunoglobulin at aconcentration of greater than or equal to 1 μg/ml were tested byindirect immunoperoxidase on tissues after the isotype of the antibody(IgA, IgG or IgM) was determined.

Polycarbonate-coated metallic beads (BioEnzaBead™, Litton Bionetics)were incubated with goat antibodies to human immunoglobulins(IgG+IgA+IgM) overnight at 4° C. and then blocked (30 min at roomtemperature) with 2.5% BSA to prevent non-specific binding. The beadswere then air dried and stored at 4° C. The ELISA for detection ofimmunoglobulin was performed as follows. Supernatant fluid from a96-well culture plate was diluted, incubated with the antibody-capturebead for 1 hr at 37° C., washed, and then incubated for 1 hr at 37° C.with peroxidase-labeled affinity-purified goat antibody to humanimmunoglobulins (IgG+IgA+IgM). The washed beads were then incubated (10min at room temperature) with2,2'-Azino-di[3-ethyl-benzthiazoline-6-sulfonic acid], and the opticaldensity was determined at 405 nm. The immunoglobulin concentrations wereinterpolated mathematically from the linear portion of a standard curve(30-1000 ng/ml) of human gamma globulin. Supernatant fluidscontaining >1 μg/ml were then isotyped using this ELISA withperoxidase-labeled goat antibodies to human γ, α, and μ chains.Subsequent quantitative assays used an immunoglobulin standardappropriate for the monoclonal antibody isotype. Mouse immunoglobulinswere assayed with Bio-EnzaBeads coated with goat antimouse IgG+IgM (H+L)and peroxidase-conjugated goat antimouse IgG+IgM (H+L). In otherexperiments, supernatant fluids were incubated with the antihuman Igbeads and the peroxidase-conjugated goat antimouse IgG+IgM (H+L).

Cryostat sections of normal and tumor tissue, stored at -30° C., werepost-fixed in PLP (0.5% p-formaldehyde, 0.075 M L-lysine, 0.01 M sodiumperiodate) for 20 minutes at 4° C. The sections were then washed.Paraffin sections of 10% formalin-fixed tissues were deparaffinizedimmediately before use. The cryostat and paraffin sections were thenincubated at room temperature in 1% bovine serum albumin in PBScontaining 0.075 M L-lysine for 20 minutes. The sections were incubatedovernight at 4° C. with hybridoma supernatant fluids. Following threewashes with PBS, the sections were then incubated with the appropriateanti-human peroxidase-labeled reagent for 60 minutes at 37° C., washedand incubated at room temperature for 15 minutes with diaminobenzidine(0.5 mg/ml, pH 7.6) in PBS containing 0.1% hydrogen peroxide. Thesections were washed with PBS, stained with hematoxyline, dehydrated,and mounted with permount.

These methods permitted the widest spectrum of tissue reactiveantibodies to be detected (i.e., directed against surface or cytoplasmicantigens).

To isolate broadly reactive antibodies, the supernatant fluids werescreened against a panel of tumor sections. Cell lines producingmonoclonal antibodies were then cloned by limiting dilution. Twenty-twofusions were performed with peripheral blood lymphocytes obtained fromten patients, and two fusions were done with lymphocytes from patientsbefore immunization. Optimal results were obtained with lymphocytesremoved one week after the second immunzation (Table 8). The frequencyof immunoglobulin producing clones isolated after the secondimmunization was almost twice that after the first immunization. Sevenof the 36 tissue-positive monoclonal antibodies reacted with cryostatsections but not with paraffin embedded tissues. This findingunderscores the need for broad screening procedures. More thantwo-thirds of the clones produced IgM, most probably a consequence ofthe source of the lymphocytes (peripheral blood).

One-third of the cell lines had morphology typical of hybridomas andgrew as dispersed cells. Karyotypic analysis of six representativehybrids demonstrated that they were human-mouse hetero-hybridomas (FIG.1A). By contrast, the majority of the monoclonal antibody synthesizingcell lines (24 out of 36) were atypical in appearance (FIG. 1B). Thesecells were predominantly irregular in shape and grew in largeaggregates. These cluster-forming cells were isolated in seven fusionsperformed with PBL from seven of ten colon patients. Thus, they appearto be quite common. Six cell lines representing five fusions from fourpatients, were karyotyped and were found to contain 46 chromosomes.G-banding of the chromosomes confirmed that they were of human origin(FIG. 1C). Thus, based upon the criterion of cell morphology, it appearsthat the majority of the monoclonal antibody-synthesizing cell lines arenot hybridomas but rather are transformed human B-cells (diploid cells).The mechanism of this spontaneous transformation is not known but may berelated to the immunization procedure.

No clear differences exist between these cell types in the isotype ofsecreted immunoglobulin or the type of tissue stained. The amounts ofimmunoglobulin (1-60 μg/ml) secreted by both cell types were essentiallycomparable, with most of the human cells producing 5-20 g/ml. As may beexpected, the diploid cells appear to be more stable with regard toimmunoglobulin production. These cells were grown in continuous culturefor up to 9 months without any indication of a finite life span forantibody production. In fact, increases in antibody production duringlong-term culture were observed for some diploid lines. The clones whichsubsequently became non-producers during extensive cell passage hadgrowth properties typical of hybridomas. However, most hybrids hadsufficient stability to permit the production of useful quantities ofantibody. For example, human-mouse heterohybridoma 7a2 was passaged formore than 20 generations from a recently cloned seed stock of 5×10⁶cells without a decrease in antibody production. Thus, the cellstheoretically could be expanded to 7×10¹³ cells. This hybrid producedapproximately 30 μg/ml/10.sup. 6 cells and thus 7×10¹³ cells couldconceivably produce over 2 kg of antibody.

D. Production of Monoclonal Antibodies

Human monoclonal antibody producing cells were grown in RPMI 1640 medium(Gibco, Grand Island, New York) supplemented with 10% fetal bovineserum, 3 mM L-glutamine and 5 μg/ml gentamicin. The medium was in somecases further supplemented with 25% D-glucose (final concentration0.25%). The cells were at 37° C. (35°-38° C.) under a humidifiedatmosphere of 7.5% CO₂ in air. The antibody was harvested from thehighly metabolized spent medium by pelletizing the medium free of cells(e.g., by centrifuging at 500 rpm for 15 minutes).

Example III: Reactivity of Monoclonal Antibodies to Normal and TumorTissue

Most of the antibodies exhibited substantially reduced binding to normalcolonic mucosa. The antibodies reactive with paraffin sections were alsotested for reactivity with normal breast, lung, gall bladder and liverand were found to be negative.

The pattern of reactivity of 10 of the human monoclonal antibodies (MCA)to histological sections of colorectal adenocarcinomas from 15 patientsis shown in FIG. 2. The matrix of reactivity of the antibodies tested,indicates that individual antibodies reacted to between 47 and 80% ofthe tumor specimens tested. No monoclonal antibodies reacted to all 15tumors. In tissue sections from individual patients, the range ofreactivity varied from tissues reactive to all 10 antibodies to tissuesreactive to as few as 1 or 2 antibodies. All of the tissue specimensused for determination of monoclonal antibody reactivity were taken frompatients other than the 10 donors of B-cells for the original fusions.

We compared the pathologic stage of the tumors tested to the percentageof reactivity with the group of monoclonal antibodies tested, and foundthat the tumors with broadest reactivity were moderately to welldifferentiated, adenocarcinomas; the less common, poorly differentiatedadenocarcinomas were generally nonreactive. The antibodies typicallyreacted with metastases.

Monoclonal antibody LiCo 16-88 reacted with an antigen preserved inparaffin-embedded sections of colorectal carcinoma that was eitherabsent or greatly reduced in normal colonic mucosa. In addition tocytoplasmic label, tumor cells exhibited surface-like staining (FIG.1D). This binding was specific, as demonstrated by the absence ofstaining by normal human immunoglobulin matched in concentration andisotype to the monoclonal antibody. Also noteworthy is the observationthat this antibody reacted with both primary tumors and metastases.Antibody LiCo 16-88 reacted with cryostat sections. As seen in FIG. 1E,intense staining of the periphery of tumor cells was observed with LiCo16-88 but not with normal human immunoglobulin (FIG. 1F).

The major advantages of a human, compared with a murine, monoclonalantibody are for in vivo diagnosis (imaging) and therapy. Less than 1%of human monoclonal antibodies isolated from tumor bearing patients werereported by previous investigators to react with cell surface antigens(Cote et al., Proc. Nat. Acad. Sci., 80:2026-2030, 1983). These findingssuggested that cancer patients may be tolerant to tumor cell surfaceantigens. It is significant, therefore, that one-half of thetissue-positive antibodies isolated from immunized patients weresubsequently found to bind to the surfaces of tumor cells (Tables 3, 4and 8). As seen in FIG. 1G, monoclonal antibody 16-88 reacts with thesurface of SW-1463 cells. The lack of staining of some of the cells maybe due to either clonal or cell cycle variations in the expression ofthe antigen(s). Thus, the greatest advantage of this invention, whichuses immunized patients as the source of sensitized B-cells, is theextremely high frequency of antibodies reactive with cell surfaceantigens produced. The antibodies produced according to the inventionhave the greatest potential for the diagnosis and treatment of cancer.

Protein (PBS and 3.0 M KCl) and lipid (chloroform-methanol) extractswere prepared from HT-29 and SW-1463 cells. Thirteen of the antibodieswere found to react with these extracts. The most striking finding wasthat all the antibodies react with the protein extracts, treatment ofthe extracts with protease significantly reduced the binding. Theseresults contrast markedly with those obtained with murine monoclonalantibodies which are often directed against glycolipid antigens of colontumors (Morgan et al., Hybridoma, 3:3, page 233, 1984), and Lindholm etal., Int. Arch. Allergy Appl. Immuno., 71:178-181, 1983).

Techniques including the preparation of protein extracts and the use ofimmunoadsorbent lectins for the immunization of mice are required toproduce monoclonal antibodies against protein antigens derived fromcolon tumors. Thus, autologous immunization of man elicits antibodiesagainst a group of antigens normally poorly immunogenic for mice. It istherefore possible that man and mice may respond to differenttumor-associated antigens. In concert with this hypothesis is thefinding that none of the 28 monoclonal antibodies examined reacted withpurified CEA, an antigen frequently seen by murine monoclonal antibodiesmade against colon tumor cells, (Koprowski et al., Somat. Cell Genet.,5:957-972, 1979, and Morgan et al., supra). It is interesting that threeof the human monoclonal antibodies also recognized antigens extracted bythe chloroform-methanol treatment. These antigens may either representproteins not denatured by this treatment or alternatively glycolipidswhich share a common epitope (i.e., the carbohydrate moiety) with aglycoprotein.

Reactivity of Human Monoclonal Antibodies to Cell Surface Antigens of 8Colon Carcinoma Cell Lines

Thirty-six human monoclonal antibodies were assessed for reactivity withtumor cell surface antigens against a panel of 8 human colon cancer celllines prepared as air-dried cytocentrifuge specimens. Thirteen of 36antibodies recognized antigens expressed on the surface of at least 2human colon carcinoma cell lines (FIG. 1H, Table 3). All 13surface-reactive antibodies were isotyped as IgM. These monoclonalantibodies were produced by both heterohybridomas and diploid B-celllines.

Experiments using murine antibodies to structural cytoplasmic antigens,such as actin, confirmed that cytoplasmic structures could not bedetected with properly prepared air-dried cytospin cell preparationswithout prior permeabilization of the cell membrane. The surfacelocalization of the antigens recognized on the Cytospinlocalizationprepared cells for most of the antibodies were confirmed by indirectimmunofluorescence of live cells.

We found no correlation between the reactivity of the monoclonalantibodies and the immunoglobulin concentration of theantibody-containing cell supernatant fluids. All cell supernatant fluidswere tested without dilution and without attempt to adjust them to aconstant immunoglobulin concentration. For the most part, the 13antibodies reactive with 2 or more cell lines exhibited more than traceactivity; the exceptions were 12-42 and 12-53, antibodies of the IgGisotype that strongly reacted to only one cell line. There was somevariation in expression of cognate antigens among the cell lines:LS-174^(t) bound to 17 monoclonal antibodies; SW-1463 and HT-29 bound to12 and 10 antibodies, respectively; the other cell lines bound to 5 to 9of the antibodies; and 7a2 and 16-52 reacted to all 8 cell lines.Otherwise, the pattern of monoclonal binding indicated a multitude ofrecognized specificities.

Reactivity of Human Monoclonal Antibodies to Cell Surface Antigenz ofDissociated Colon Carcinoma Tumor Cells

We confirmed the cell surface reactivity observed with the colon celllines in assays on air-dried Cytospin preparations of enzymaticallydissociated colon tumor cells from 9 patients (Table 4). Seventeen ofthe monclonal antibodies reacted to at least 2 of the tumor cellpreparations. There were some differences between the cell line data andthe tumor cell data; 16-86, which reacted with 4 out of 8 cell lines,gave positive results with only one tumor cell preparation, and 16-105and 12-53, which reacted with 0 out of 8 and 1 out of 8 colon celllines, respectively, reacted with 3 or more of the tumor cellpreparations. As was seen from the assays of reactivity with cell lines,the patterns of antibody binding, which reflect the presence and degreeof antigen expression by the tumor cells, suggest that many differentspecificities are recognized by these monoclonal antibodies.

Reactivity of Human Monoclonal Antibodies with Paraffin Sections ofPaired Colon Tumor and Normal Mucosa

The specificity of 25 of the human monoclonal antibodies reactive withparaffin sections was tested by indirect immunohistochemistry againstpaired sections of colonic tumor and autologous normal colonic mucosafrom 5 patients (Table 5). Eleven of the 25 (44%) demonstrated nodetectable reactivity with normal colonic mucosa in the 5 patientstested, but all 11 reacted with tumor specimens. Fourteen of the 25antibodies, although reactive with the tumor specimens, also reactedwith normal colonic mucosa. Quantitatively, in these cases reactivitywith normal colonic specimens was less than with tumor specimens.Individual antibodies reacted with 1 to 4 of the normal colonic mucosaspecimens tested. Five of 14 of these cross reactive antibodies onlyreacted with the normal colonic mucosa of 1 of the 5 patients. Thenormal colonic mucosa of patient 8 reacted with 13 of the 23 antibodiesthat reacted with that patient's tumor. Whether the normal colonicmucosa from this patient was proximal or distal to the tumor is notknown. If patient 8 were eliminated from this analysis only 9 of 24antibodies tested would have reacted with 1-3 of the normal colonicmucosa paired samples from 5 patients. Overall, in the total pairedcolorectal tumor and normal colonic mucosa specimens tested,approximately 30% showed cross reactivity with normal colonic mucosa wasseen, although the quantitative reactivity was significantly less thanthat observed against the paired tumor specimen. Moreover, theoccurrence of a lower level but detectable normal cell reactivity may beattributable to the recognition determinants associated with a deviationfrom the normal conditions which does not show as cancerous.

Reactivity of Human Monoclonal Antibodies with Paired Human Colon Tumorand Mucosa Cell Cytospin Preparations by Direct Binding ofBiotin-Labeled Antibodies

The specificity of antibodies for tumor cells versus normal cells isdifficult to evaluate by indirect staining methods on Cytospinpreparations and cryostat sections. The peroxidase-labeled antihuman Igantibodies used to detect the human antibodies also recognize endogenoushuman immunoglobulin present on all human tissues. Normal tissuescontain greater amounts of endogenous immunoglobulin than docorresponding tumor tissues, consequently the background is higher fornormal than for tumor tissue. Direct labeling of the antibodiesovercomes this problem and permits inclusion of an excess of irrelevanthuman immunoglobulin with the monoclonal antibodies to block nonspecificimmunoglobulin binding, another problem associated with indirecttechniques.

Five of the surface-reactive human antibodies were purified from culturemedium and labeled wth biotin. The 5 were chosen because they hadreacted well in previous assays and produced relatively high levels ofhuman immunoglobulin. Table 6 shows the results with the 5biotin-labeled antibodies in direct assays on air-dried Cytospin cellpreparations of colon tumor and adjacent mucosa cells obtained from 7patients. All 5 antibodies reacted with the tumor cells, confirming thereactivity seen in indirect assays. Reactivity with normal mucosa cellswas weak or non-detectable.

Direct Binding of Biotin-Labeled Monoclonal Antibodies to Frozen TissueSections of Colon Tumor and Normal Colonic Mucosa

Further direct characterization of the 5 biotin-labeled antibodies withregard to their specificity for tumor versus normal cells wasestablished with frozen tissue sections of colon tumor and adjacentnormal colonic mucosa (Table 7). Absolute specificity was observed with4 of the antibodies as shown by the fact that they strongly reacted withat least 2 out of 5 colon tumors and did not react with any of the 4matched normal colonic mucosa sections. 19b2 reacted strongly with 4 of5 tumor sections and showed a weak reaction with 1 of 4 normal colonicmucosa sections. 19b2 also reacted somewhat with normal colonic mucosaCytospin cell preparations (Table 6) and normal colonic mucosa paraffinsections (Table 5).

Frozen tissue sections of normal breast, stomach, kidney, liver, muscleand skin (Table 7) showed no staining by biotin-labeled human antibodiesexcept antibody 19b2 which exhibited a low level of binding to normalstomach tissue. An overall background stain of connective tissuecomponents was observed. This background staining was nonspecific andhas been observed by others using biotin-labeled monoclonal antibodies.

Reactivity of Monoclonal Antibodies with CEA, Erythrocyte and LeukocyteAntigens

To further establish the tumor specificity of the monoclonal antibodies,we tested for reactivity with CEA, human erythrocyte antigens and humanlymphocyte antigens by various techniques. We found no evidence orreactivity between these antibodies and these antigens. Anti-CEAactivity was assessed by ELISA against two CEA preparations. Thestaining patterns of the human monoclonal antibodies on human colontumor paraffin sections were different from those observed with a mouseanti-CEA antibody. None of the 36 human antibodies gave the luminalstaining pattern typically seen with anti-CEA antibodies. Reactivitywith human erythrocyte antigens was measured by indirectimmunofluorescence and hemagglutination against an erythrocyte panelrepresenting all major and most minor blood group systems. No reactivitywas seen. ELISA, cytotoxicity assays and indirect immunoperoxidasestaining of human lymphocytes showed no evidence of recognition of humanlymphocyte antigens by any of the antibodies.

Functionality of Human Monoclonal Antibodies to Colorectal Cancer

Specificity is a major consideration in the determination of theusefulness of these tumor-reactive monoclonal antibodies. The lack ofreactivity of some of the monoclonal antibodies with a certainpercentage of the tumor specimens tested is another factor which must beconsidered. Thus it is unlikely, based upon these data, that any singlemonoclonal antibodies would have all the factors associated with it thatwould make it ideal for therapeutic or diagnostic application. Thestrategy of using immunized cancer patients has provided a large numberof clones from which certain selections can be made with regard to rangeof reactivities, as well as specificity. By selecting only 2 of themonoclonal antibodies that we have produced which, based on theircharacteristics in a broad in vitro screen, have the greatest amount oftumor reactivity with the least amount of normal colonic mucosareactivity, we can propose and develop cocktails of antibodies thattogether promise greater efficacy than any individual monoclonalantibody. As shown in FIG. 3, 2 monoclonal antibodies, 6a3-1 and 7a2,paired for their range of reactivity with both tissue sections anddissociated tumor cells and selected based on their relative lack ofcross reactivity with normal colonic mucosa, provide an antibodycocktail which will react with 14 of 15 tumor specimens and 9 of 9dissociated tumor cell specimens. Other cocktails of this type can bedeveloped; however, clearly we must have a broad range of monoclonalantibodies to select from and an extensive in vitro screen for testing alarge number of specimens in a variety of differentiation states inorder to utilize human monoclonal antibodies for therapeutic ordiagnostic purposes.

In addition to providing monoclonal antibodies reactive with tumor cellsurface antigens for the in vivo diagnosis and immunotherapy of cancer,the invention provides monoclonal antibodies which will be useful asprobes to isolate and characterize the antigens relevant to human cancerimmunity. These antigens may ultimately prove useful as a tumor vaccine.In addition, the generation of antibody producing diploid cells adds adimension of genetic stability to the production of human monoclonalantibodies reactive with tumor cell surface antigens.

Table 3 shows the tissue reactivity of monoclonal antibodies produced bythe monoclonal antibody cell lines prepared according to theseprocedures.

The foregoing describes the formation of novel monoclonal antibodiesspecific for certain tumors, hybridomas, and methods for theirpreparation. The techniques for preparing the novel monoclonalantibodies, hybridomas, and diploid cells have been described in detail,particularly with reference to specific embodiments included by way ofthe examples. It will be understood that the products and techniques ofthe present invention are of far-reaching significance in the field ofcancer detection and treatment. They include a wide range of monoclonalantibodies, each specific for determinants found on an individual strainof tumor forming cancer, as the technique disclosed herein can be usedto generate antibodies for every such case. It will be furtherunderstood that many variations and modifications of the techniquesdisclosed herein are available to those of ordinary skill in therelevant art and that such variations and modifications are contemplatedas being within the scope of the invention.

The embodiments provided to illustrate this invention relate tocarcinoma tumors, particularly well-differentiated colorectaladenocarcinomas. Clearly, however, the invention pertains to allcarcinomas, such as lung, breast, and other malignancies in areas whicharise from the same type of embryonic tissue. Moreover, the proceduresdescribed can be adjusted, if necessary, by one skilled in the art to beused to apply this invention to other types of cancer.

                  TABLE 1                                                         ______________________________________                                        Criteria for Successful Vaccines for                                          Active Specific Immunotherapy                                                 ______________________________________                                        Adjuvant                                                                      (a)    BCG (Phipps, Tice, Connaught); lyophilized,                                   frozen (dose-dependence >10.sup.6 (10.sup.7 -10.sup.8)                 (b)    C. parvum (Wellcome Labs) (dose-dependence >                                  7 μg (70 μg-700 μg)                                           Tumor Cells                                                                   (a)    Enzymatic dissociation                                                 (1)      Collagenase type I (1.5-2.0 U/ml HBSS)                               (2)      DNAase (450 D.U./ml HBSS)                                            (3)      37° C. with stirring                                          (b)    Cryopreservation                                                       (1)      Controlled-rate freezing (-1° C./min) (7.5%                            DMSO, 5% HSA, HBSS)                                                  (2)      Viability 80%                                                        (c)    X-irradiation                                                          (1)      Rendered non-tumorigenic at 12,000-20,000 R.                         Components and Administration.sup.a                                           (a)    Ratio of adjuvant to tumor cells - 10:1-1:1                                   (optimum)                                                              (b)    10.sup.7 tumor cells (optimum)                                         (c)    2-3 i.d. vaccinations at weekly intervals.                                    Third vaccination contains tumor cells only.                           ______________________________________                                         .sup.a Isoniazid chemoprophylaxis of BCG infection optional.                  BCG  Bacillus Calmette Guerin                                                 HBSS  Hanks' balanced saline solution                                         DMSO  Dimethylsulfoxide                                                       HSA  Human serum albumin                                                      R  Rads                                                                       PBS  Phosphate buffered saline                                                EDTA  Ethylenediaminetetraacetic acid                                    

                  TABLE 2                                                         ______________________________________                                        DCH Reaction to Autologous Tumor Cells                                                                           Reactivity                                                        Pre-        6 wk                                                     No. of   immunization                                                                              and/or                                             Stage Patients Reactivity.sup.a                                                                          6 mo.                                      ______________________________________                                        Immunized B2      8        0         4                                        Patients:                                                                               C1,C2   9        2         6                                                  D       7        2         6                                        Total (%)         24       4 (17%)   16 (67%)                                 Nonimmunized                                                                            B2      4        1         0                                        Patients:                                                                               C1,C2   5        0         1                                                  D       2        0         0                                        Total (%)         11       1 (9%)     1 (9%)                                  ______________________________________                                         .sup.a Reactions were considered positive when the 48hr. induration (the      mean of 2 diameters) was more than 5 mm.                                 

                                      TABLE 3                                     __________________________________________________________________________    Reactivity of Human Monoclonal Antibodies to Cell Surface Antigens            of Eight Colon Carcinoma Cell Lines.sup.a                                            Colon Carcinoma Cell Lines                                             Monoclonal                                                                    Antibody                                                                             Concentration.sup.b                                                                   Isotype                                                                           HT-29                                                                             SW1463                                                                             SW948                                                                             SW480                                                                             SW403                                                                             LS-174.sup.t                                                                       LoVo                                                                              WiDr                         __________________________________________________________________________    6a3*.sup.c                                                                           11      IgM 2+  +    -   -   -   4+   +   +                            7a2*   23      IgM 4+  4+   4+  4+  4+  4+   4+  4+                           7a4*   18      IgM +   4+   -   2+  -   3+    +  -                            11A7    3      IgM -   -    -   -   -   -    -   -                            11B5    7      IgM -   -    -   -   -   2+   -   -                            12-38* 144     IgG -   -    -   -   -   -    -   -                            12-42* 74      IgG -   -    -   -   -   -    3+  -                            12-47* 25      IgG -   -    -   -   -   -    +   -                            12-53* 219     IgG -   -    -   -   -   -    -   2+                           15-12  15      IgM -   -    -   -   -   -    -   +                            15-24  18      IgG -   ND   ND  -   -   -    -   -                            15-33  11      IgM -   -    -   -   -   +    -   -                            15-39   3      IgG -   -    -   -   -   -    -   -                            16-4   19      IgM -   -    -   -   -   +    +   -                            16-50   3      IgM -   ND   ND  -   -   +    ND  ND                           16-52   4      IgM 2+  3+   +   3+  3+  3+   4+  3+                           16-58  14      IgM 3+  2+   -   4+  2+  2+   +   -                            16-66   7      IgM +   2+   -   4+  3+  +    +   -                            16-72   5      IgM -   -    -   -   -   -    -   -                            16-80   8      IgG -   -    -   -   -   -    -   -                            16-81   6      IgM -   -    -   -   -   -    -   -                            16-86   9      IgM -   3+   -   -   -   4+   4+  4+                           16-88   9      IgM 3+  4+   3+  -   3+  4+   4+  4+                           16-103  6      IgM -   -    -   -   -   +    -   -                            16-105 11      IgM -   -    -   ND  -   -    -   -                            18-15  16      IgG -   -    -   +   -   -    -   -                            18-21* 12      IgM 2+  2+   -   -   -   2+   -   -                            18-22*  7      IgM +   +    +   -   +   -    2+  +                            19b2*  26      IgM 3+  4+   2+  -   +   4+   4+  4+                           20A3    4      IgG -   -    -   -   -   +    -   -                            20A6    9      IgG -   -    -   -   -   -    -   -                            20B7    9      IgG -   -    -   -   -   -    -   -                            21B27* 19      IgM -   -    -   -   -   -    -   -                            23A4    8      IgM -   -    ND  ND  ND  -    ND  ND                           27B1    3      IgM -   -    ND  ND  ND  -    ND  ND                           28A32*  3      IgM -   2+   ND  ND  ND  2+   ND  ND                           __________________________________________________________________________     .sup.a Intensity of immunoperoxidase staining compared to the control         matched in isotype and concentration to the monoclonal antibody tested.       .sup.b μg /ml.                                                             .sup.c "*" Designates hybridomas culture morphology, other appeared as        transformed (diploid) B cell lines.                                           Cell lines. Human colonic adenocarcinoma cell lines HT29, SW1463, SW948,      SW480, SW403, LoVo, and WiDr were obtained from the American Type Culture     Collection (Rockville, Maryland). The cells were cultured in the              recommened culture medium supplemented with 10% fetal bovine serum. Colon     adenocarcinoma cell line LS174.sup.t obtained from Dr. Jeffery Schlom,        (National Cancer Institute, Bethesda, Maryland), was cultured in              Dulbecco's modified Eagle's medium. All Cell lines were incubated at          37° C. in an atmosphere of 5% CO.sub.2.                           

                                      TABLE 4                                     __________________________________________________________________________    Reactivity of Human Monoclonal Antibodies to Cell Surface                     Antigens of Colon Carcinoma Tumor Cells.sup.a                                 Monoclonal         Patient Number                                             Antibodies                                                                           Concentration                                                                         Isotype                                                                           16 17 18 19 20 21 22 23 24                                 __________________________________________________________________________    6a3*.sup.c                                                                           11      IgM +  -  -  -  2+ 3+ +  -  -                                  7a2*   23      IgM 4+ 4+ 4+ 4+ 4+ 4+ -  +  4+                                 7a4*   18      IgM 2+ -  2+ 2+ -  4+ ND -  -                                  11B5    7      IgM -  -  -  -  -  +  -  -  ND                                 12-38* 144     IgG -  -  -  -  -  -  -  -  ND                                 12-42* 74      IgG -  -  -  -  -  -  -  -  ND                                 12-47* 25      IgG -  -  -  -  -  -  ND ND ND                                 12-53* 219     IgG 2+ -  3+ ND -  -  -  -  2+                                 15-12  15      IgM -  -  -  -  +  +  -  -  -                                  15-24  18      IgG -  -  -  -  -  -  ND -  ND                                 15-33  11      IgM -  -  -  -  +  2+ -  -  -                                  15-39   3      IgG -  -  -  -  -  -  -  -  ND                                 16-4   19      IgM -  -  -  -  -  3+ -  -  ND                                 16-50   3      IgM -  -  -  -  -  +  ND -  ND                                 16-52   4      IgM 2+ 2+ 2+ 2+ +  2+ +  +  -                                  16-58  14      IgM 3+ 3+ 3+ 2+ -  +  -  -  3+                                 16-66   7      IgM 4+ 4+ 3+ -  +  -  -  -  -                                  16-72   5      IgM -  -  -  -  -  2+ -  -  -                                  16-80   8      IgG -  -  -  -  +  -  +  -  -                                  16-81   6      IgM -  +  -  -  +  -  -  -  ND                                 16-86   9      IgM -  -  -  -  +  -  -  -  -                                  16-88   4      IgM +  +  +  3+ 4+ -  -  +  -                                  16-103  6      IgM -  -  -  -  -  -  ND -  ND                                 16-105 11      IgM -  -  2+ +  +  +  ND ND ND                                 18-15  16      IgG -  +  -  -  -  +  -  -  ND                                 18-21* 12      IgM +  +  +  +  -  +  -  -  -                                  18-22*  7      IgM 2+ +  2+ 2+ +  2+ -  +  -                                  19b2*  26      IgM 2+ 2+ 2+ 2+ 3+ 4+ +  2+ -                                  20A3    4      IgG -  -  -  -  -  +  -  -  ND                                 20A6    9      IgG -  -  -  -  -  -  -  -  ND                                 20B7    9      IgG -  +  -  -  -  -  -  -  ND                                 21B27* 19      IgM -  -  -  -  -  -  -  -  ND                                 __________________________________________________________________________     .sup.a The presence and degree of binding are as explained in the             footnotes to Table 3.                                                         .sup.b μg/ml.                                                              .sup.c * Designates hybridoma culture morphology, other grow as               transformed (diploid) Bcell lines.                                       

                                      TABLE 5                                     __________________________________________________________________________    Reactivity of Human Monoclonal Antibodies on Paraffin Sections                of Colorectal Tumors (T) and Paired Normal Colonic Mucosa (N).sup.a                  Patient Number                                                         Monoclonal                                                                           2     6     7     8     10                                             Antibodies                                                                           T  N  T  N  T  N  T  N  T  N                                           __________________________________________________________________________    6a3*   +  -  -  nd 2+ -  2+ -  2+ -                                           7a2*   -  ND 3+ -  -  ND 4+ -  -  ND                                          7a4    -  ND -  ND -  ND 4+ -  -  ND                                          11B5   +  -  3+ -  3+ -  4+ 2+ 3+ +                                           12-38  -  ND -  -  2+ -  2+ -  3+ -                                           12-42  -  ND -  ND 2+ -  3+ -  2+ -                                           12-47  +  -  -  ND +  -  2+ -  -  ND                                          12-53  -  ND 3+ -  -  ND +  -  -  ND                                          15-24-2                                                                              -  ND 3+ -  4+ 2+ 3+ +  -  ND                                          16-4   -  ND 2+ -  +  -  3+ +  +  -                                           16-58  -  ND 4+ +  4+ -  2+ +  -  ND                                          16-66  -  ND 4+ -  +  +  3+ +  +  -                                           16-86  -  ND -  ND 2+ -  +  -  +  -                                           16-88  +  -  2+ -  +  -  4+ +  +  -                                           18-15  +  -  2+ -  +  -  2+ +  +  -                                           18-21  -  ND 3+ +  2+ +  3+ +  +  +                                           18-22  -  ND -  ND +  -  -  ND +  -                                           19b2   -  ND -  ND 2+ +4+                                                                              2+ -  ND                                             20A3   +  -  4+ +  +  -  2+ -  +  -                                           20A6   3+ -  2+ -  2+ -  2+ -  2+ +                                           20B7   3+ +  2+ -  +  -  3+ +  +  +                                           21B27  2+ +  2+ -  -  ND 3+ +  -  ND                                          23A4   -  ND 2+ -  2+ -  2+ +  3+ -                                           27B1   2+ -  4+ +  3+ -  4+ 2+ 3+ +                                           28A32  -  ND 4+ -  +  -  -  ND +  -                                           __________________________________________________________________________     .sup.a Presence and degree of binding are indicated as explained in the       footnotes of Table 3.                                                    

                                      TABLE 6                                     __________________________________________________________________________    Reactivity of Biotin-Labeled Monoclonal Antibodies to Human Colon             Tumor (T) and Normal Mucosa Cell (N) Cytospin Preparations.sup.a                      Monoclonal Antibodies                                                         6a3   7a2   7a4     18-22                                                                             19b2                                          Patient Number                                                                        T  N  T  N  T  N  T  N  T  N                                          __________________________________________________________________________    18      +  -  +  +  +  +  +  +  +  +                                          21      3+ -  +  +  2+ -  +  +  +  -                                          24      2+ -  +  +  4+ -  2+ -  3+ -                                          25      +  -  +  -  +  +  2+ 2+ +  +                                          26      -  -  -  -  +  +  -  -  +  -                                          27      2+ -  -  -  2+ -  2+ +  2+ 2+                                         28      +  +  +  -  +  +  +  -  2+ +                                          __________________________________________________________________________     .sup.a The presence and degree of binding are indicated as explained in       the footnote to Table 3.                                                 

                                      TABLE 7                                     __________________________________________________________________________    Reactivity of Biotin-Labeled Monoclonal Antibodies with Frozen                Section of Colon Tumors (T) and Normal Tissues (N).sup.a                               Monoclonal Antibodies                                                         6a3   7a2   7a4   18-22 19b2                                         Source of Tissue                                                                       T  N  T  N  T  N  T  N  T  N                                         __________________________________________________________________________    Colon    +  -  -  -  2+ -  +  -  2+ +                                         Colon    3+ -  2+ -  3+ -  +  -  2+ -                                         Colon    3+ -  +  -  3+ -  3+ -  3+ -                                         Colon    2+ -  +  -  -  -  -  -  -  -                                         Breast      -     -     -     -     -                                         Breast      -     -     -     -     -                                         Breast   -  -  -  -  -  -  -  -  -                                            Stomach     -     -     -     -     +                                         Kidney      -     -     -     -     -                                         Liver       -     -     -     -     -                                         Muscle      -     -     -     -     -                                         Skin        -     -     -     -     -                                         Skin        -     -     -     -     -                                         __________________________________________________________________________     .sup.a The presence and degree of binding are indicated as explained in       the footnotes to Table 3.                                                

                                      TABLE 8                                     __________________________________________________________________________    Isolation of Human Monoclonal Antibodies Reactive with Colorectal             Carcinoma                                                                             No. of                                                                             Wells                                                                              No. of Ig.sup.+                                                                       No. of Tissue.sup.+                                                                   No. of Cell Surface.sup.+                                                                Isotype.sup.d                                                                       Culture Pattern.sup.e      Immunization.sup.a                                                                    Patients                                                                           Assayed                                                                            Cell Lines (%).sup.b                                                                  Cell Lines                                                                            Cell Lines (%).sup.c                                                                     IgG                                                                              IgM                                                                              Diploid                                                                             Hydridoma            __________________________________________________________________________    Pre     2     25  4       0   (0%)                                                                              0    (0%)  0  0                             1       9    441  65  (15%)                                                                             10/65                                                                             (15%)                                                                             4/10 (40%) 2  8   8    2                    2       10   573  154 (27%)                                                                             25/154                                                                            (9%)                                                                              16/25                                                                              (64%) 9  16 16    9                    3       3    112  11  (10%)                                                                             1/11                                                                              (10%)                                                                             0/1  (0%)     1        1                    __________________________________________________________________________     .sup.a PBL were obtained 7 days after each immunization.                      .sup.b Production of ≧1 μg/ml of human immunoglobulin as            measured by ELISA.                                                            .sup.c Immunoperoxidase label of unfixed airdried preparations of SW1463,     HT29 or enzymatically dissociated tumor cells.                                .sup.d Isotypes were determined by ELISA.                                     .sup. e Diploid cells, cell growth in clusters. Hybridoma cells, growth a     dispersed cells.                                                         

We claim:
 1. A method for preparing a monoclonal antibody thatspecifically binds to tumor-associated antigens, comprising exposing invivo a human B-lymphocyte to a human tumor cell antigen in a vaccinecomprising autologous viable human tumor cells that have been madenon-tumorigenic to activate the B-lymphocyte, contacting said activatedB-lymphocyte with a myeloma cell under conditions suitable for fusion toproduce a fused B-lymphocyte, culturing the fused B-lymphocyte underconditions suitable for expression of antibodies and recovering saidmonoclonal antibody.
 2. A method for preparing a hybridoma cell thatproduces a human monoclonal antibody that specifically binds to tumorassociated antigens, comprising exposing in vivo a human B-lymphocyte toa human tumor cell antigen in a vaccine comprising autologous viablehuman tumor cells that have been made non-tumorigenic to activate theB-lymphocyte, contacting the activated B-lymphocyte with a myeloma cellunder conditions suitable for fusion and recovering the hybridoma cellfusion product.
 3. The method of claim 2, wherein said B-lymphocytes areactivated by the process comprising immunizing a human subject withviable, non-tumorigenic, autologous tumor cells from the subject's owntumor.
 4. The method of claim 2 for making hybridoma cells that producehuman monoclonal antibodies that specifically bind to tumor associatedantigens, comprising:(a) surgically removing tumor tissue from a cancerpatient; (b) digesting the tumor tissue to obtain tumor cells; (c)deactivating the tumor cells to prepare a viable but non-tumorigenictumor cell preparation; (d) preparing a vaccine from the viablenon-tumorigenic cell preparation; (e) injecting the vaccine into asubject; (f) collecting peripheral blood from the subject; (g) obtainingB-lymphocyte cells from the peripheral blood of the subject; (h) mixingthe B-lymphocytes with myeloma cells under hybridizing conditions toproduce hybridoma cells; (i) culturing the hybridoma cells; (j)screening the hybridoma cell cultures for the production of humanimmunoglobulin; and (k) screening the hybridoma cell cultures thatproduce human immunoglobulin for anti-tumor antibody activity.
 5. Themethod of claim 4, wherein the vaccine comprises approximately 10⁷viable tumor cells.
 6. A method for making cells that produce humanmonoclonal antibodies that specifically bind to tumor associatedantigens, comprising:(a) surgically removing tumor tissue from a cancerpatient; (b) digesting the tumor tissue to obtain tumor cells; (c)deactivating the tumor cells to prepare a viable but non-tumorigenictumor cell preparation; (d) preparing a vaccine from the viablenon-tumorigenic cell preparation; (e) injecting the vaccine into asubject; (f) collecting B-lymphocyte cells from the subject; (g)culturing the lymphocyte cells; (h) screening the lymphocyte cellcultures for cluster formation; (i) screening the clustered cellcultures for the production of human immunoglobulin; and (j) screeningthe clustered cell cultures that produce human immunoglobulin foranti-tumor antibody activity.
 7. A method for preparing a monoclonalantibody that specifically binds to tumor associated antigens comprisingculturing a clustered cell culture according to claim 6 and recoveringhuman immunoglobulin produced by said culture.